Department of Biotechnology, School of Life Sciences, University of Hyderabad, Hyderabad, India.
J Phys Chem B. 2011 Dec 15;115(49):14760-7. doi: 10.1021/jp2062496. Epub 2011 Nov 15.
Protein-surfactant interaction is widely studied to understand stability and structural changes in proteins. In this Article, we have investigated SDS-induced unfolding of RNase A using absorbance, intrinsic fluorescence of the protein, anisotropy, TNS fluorescence, and near- and far-UV circular dichroism. Unfolding titration curves obtained from the absorbance and fluorescence changes were fitted into a five-state protein unfolding model by assuming formation of three intermediate states. Free energy changes and m-values of all four transitions between the native and unfolded state were evaluated. The transitions are categorized into two different regions. Region I, up to 0.5 mM of SDS, involves ionic interaction between the protein and SDS where the secondary and tertiary structure of the protein is altered to a less extent. In region II, hydrophobic interaction dominates and has two distinct transitions. The first transition arises from the aggregation of surfactant molecules around the protein hydrophobic sites. In the following transition, the micelles probably expand more, and a few more hydrophobic sites are occupied by the surfactant. In this region, the tertiary contacts are completely broken, and almost 50% of the secondary structure is lost. The aggregation of SDS around the protein starts well below the CMC. These conformational changes can be explained by the necklace and beads model, and the free energy of formation of such a complex for the RNase A-SDS system is found to be 5.2 (±1.0) kcal mol(-1). The probable interaction sites and the mechanism of unfolding have been discussed in detail.
蛋白质-表面活性剂相互作用被广泛研究，以了解蛋白质的稳定性和结构变化。在本文中，我们使用吸光度、蛋白质的固有荧光、各向异性、TNS 荧光以及近紫外和远紫外圆二色性研究了 SDS 诱导的 RNase A 变性。通过假设形成三个中间态，将从吸光度和荧光变化获得的展开滴定曲线拟合到五态蛋白质展开模型中。评估了所有四个从天然态到展开态的转变之间的自由能变化和 m 值。这些转变分为两个不同的区域。区域 I，直到 SDS 浓度为 0.5 mM，涉及蛋白质与 SDS 之间的离子相互作用，其中蛋白质的二级和三级结构变化程度较小。在区域 II 中，疏水相互作用占主导地位，并有两个明显的转变。第一个转变是由于表面活性剂分子在蛋白质疏水部位周围聚集。在随后的转变中，胶束可能会进一步扩张，更多的疏水部位被表面活性剂占据。在这个区域，三级接触完全断裂，几乎 50%的二级结构丢失。SDS 围绕蛋白质的聚集在 CMC 以下就开始了。这些构象变化可以用项链和珠子模型来解释，并且发现 RNase A-SDS 体系形成这种复合物的自由能为 5.2（±1.0）kcal/mol。详细讨论了可能的相互作用位点和展开机制。
